南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (09): 1065-.doi: 10.12122/j.issn.1673-4254.2019.09.10

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人卵巢癌SKOV3 细胞筛选获得的肿瘤干细胞样细胞在血管生成拟态形成中的作用

梁军，邢慧敏，吴小华，张蕾，赵郡   

  • 出版日期:2019-09-20 发布日期:2019-09-20

Role of ovarian tumor stem-like cells sorted from human epithelial ovarian cancer SKOV3 cells in vasculogenic mimicry formation

  • Online:2019-09-20 Published:2019-09-20

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摘要： 目的探讨卵巢癌SKOV3细胞中分离肿瘤干细胞样细胞的生物学特性及其在血管生成拟态（VM）形成中的作用。方法 采用无血清悬浮培养法对SKOV3细胞培养传代至第7代；流式细胞术测定亲代SKOV3细胞及第1、3、5、7代分选细胞CD133、 CD117阳性细胞比例；平板克隆形成和小鼠体内成瘤实验鉴定分选细胞（第7代，下同）生物学特性；三维立体培养比较分选细 胞与亲代SKOV3细胞形成VM的能力。采用实时荧光定量聚合酶链式反应(RT-PCR)方法及Western blot法检测分选细胞与亲 代SKOV3细胞的基质金属蛋白酶2，9的表达水平。结果SKOV3部分细胞能在无血清培养基中形成悬浮生长的细胞球，可连 续传代至第7代；流式细胞术结果显示CD133、CD117阳性细胞比例随传代递增。分选细胞克隆形成率高于亲代SKOV3细胞 （P<0.01）。小鼠体内成瘤实验显示第7代悬浮细胞的成瘤率（6/6）明显高于亲代细胞的成瘤率（0/6）。三维立体培养发现分选 细胞比亲代SKOV3细胞VM形成能力强。第7代分选细胞基质金属蛋白酶2，9基因和蛋白表达较SKOV3细胞均增高，差异均 具有统计学意义（P<0.01）。结论采用无血清悬浮培养法培养的SKOV3分选细胞具有肿瘤干细胞的生物学特性，具有较强的 VM形成能力，可能在VM形成中发挥作用。

Abstract: Objective To isolate tumor stem-like cells from human epithelial ovarian cancer SKOV3 cells and explore their role in the formation of vascularization mimicry (VM). Methods SKOV3 cells were passaged to the 7th generation by suspension culture in serum-free medium, and the percentages of CD133- and CD117-positive cells in the 1st, 3rd, 5th and 7th generations were analyzed using flow cytometry. The proliferative activity of the cells sorted from the 7th generation SKOV3 cells was assessed with colony formation assay. A three-dimensional cell culture model was established to compare the ability of VM formation between the sorted cells and the parental SKOV3 cells. The expression levels of matrix metalloproteinases-2 (MMP-2) and MMP-9 in the two groups were detected using real-time PCR and Western blotting. Results Some SKOV3 cells formed typical cell spheres with suspension growth in serum-free medium and were passaged to the 7th generation. Flow cytometry revealed that the percentage of CD133-positive cells increased with cell passaging. The cloning efficiency of the sorted cells was significantly higher than that of the parental SKOV3 cells (50.33% vs 5.33%, P<0.001). The VM formation ability of the sorted cells was stronger than that of the parental SKOV3 cells in the three-dimensional cell culture system. RT-PCR and Western blotting showed that the expression levels of MMP-2 and MMP-9 were significantly higher in the 7th passage cells than in the parental cells (P<0.05). Conclusion The sorted cells from SKOV3 cells cultured in serum-free medium exhibit biological properties of tumor stem cells with strong VM formation ability, suggesting their role in VM formation.